Reinforced tissue graft

ABSTRACT

A biocompatible tissue graft is provided. The tissue graft includes an extracellular matrix patch and a means for reinforcing the patch.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.61/040,066, filed Mar. 27, 2008, the subject matter, which isincorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention is directed to tissue grafts and, in particular,is directed to a reinforced tissue graft.

BACKGROUND OF THE INVENTION

Current treatment for rotator-cuff tears is to suture the torn tendonback to the bone of the humoral head. The sutures hold the tendon incontact with the bone, preferably long enough for the tendon to heal tothe bone and form a bridge that will re-establish the tendon-boneconnection and restore normal function. The sutures that are usedpossess sufficient tensile strength to retain the tendon and bonetogether during the healing process. However, the tendon is a fibroustissue that can be torn by the sutures. The sutures can align with thefascicular structure of the tendon and tear through it under sufficienttensile force undoing the surgical repair before tendon-to-bone healingis complete. The sutures can also tear through the bone under sufficientforce, particularly in older subjects who form the bulk ofrotator-cuff-tear patients and whose bones tend to be more osteoporotic.

SUMMARY OF THE INVENTION

The present invention relates to a reinforced, biocompatible tissuegraft. The tissue graft includes an extracellular matrix patch (ECM) anda means for reinforcing the graft to mitigate tearing of the graftand/or to improve the fixation retention of the graft when fixed orsecured to tissue being treated. The reinforcing means can include afiber stitched into the ECM patch in a reinforcement pattern. The fibercan be formed from a biocompatible material and have a high modulus ofelasticity and failure load. Examples of biocompatible materials thatcan be used to form the fiber include silk, sericin-free silk, modifiedsilk fibroin, polyesters, such as poly(glycolic acid) (PGA), poly(lacticacid) (PLA), poly(ethylene glycol) (PEG), polyhydroxyalkanoates (PHA)and polyethylene terephthalate (PET), medical grade polyethylene, suchas polyethylene (UHMWPE), blends thereof and copolymers thereof, as wellas other biocompatible materials that are typically used in formingbiocompatible fibers for in vivo medical applications.

Another aspect of the present invention relates to a biocompatibletissue graft that includes a fascia patch and at least one fiberstitched into the patch in a reinforcement pattern to mitigate tearingand/or improve fixation retention of the patch.

Yet another aspect of the present invention relates to a method ofconstructing a biocompatible tissue graft that includes providing anextracellular matrix patch and stitching at least one fiber into thepatch in a reinforcement pattern to mitigate tearing and/or improvefixation retention of the patch.

Still a further aspect of the present invention relates to a method forrepairing tissue in a subject that includes administering to the tissuea biodegradable tissue graft. The biocompatible tissue graft includes anextracellular matrix patch and at least one fiber stitched into thepatch to mitigate tearing and/or improve fixation retention of thepatch.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill become apparent to those skilled in the art to which the presentinvention relates upon reading the following description with referenceto the accompanying drawings, in which:

FIG. 1 is schematic illustration of a tissue graft having reinforcementmeans in accordance with an embodiment of the present invention;

FIG. 2 is a top view of a tissue graft having a reinforcement means inaccordance with another embodiment of the present invention;

FIG. 3 a is a top view of a tissue graft having a reinforcement means inaccordance with yet another embodiment of the present invention;

FIG. 3 b is a top view of a tissue graft having a reinforcement means inaccordance with yet another embodiment of the present invention;

FIG. 3 c is a top view of a tissue graft having a reinforcement means inaccordance with yet another embodiment of the present invention;

FIG. 4 a is a schematic illustration of a tissue graft having areinforcement means in accordance with yet another embodiment of thepresent invention;

FIG. 4 b is a top view of a tissue graft having a reinforcement means inaccordance with yet another embodiment of the present invention;

FIG. 5 a is a schematic illustration of a fiber of a tissue graft inaccordance with yet another embodiment of the present invention;

FIG. 5 b is a graph illustrating the uniaxial suture retention strengthof unreinforced and reinforced tissue grafts;

FIG. 6 is a graph illustrating the multi-directional suture retentionstrength of unreinforced and reinforced tissue grafts;

FIG. 7 is a graph illustrating the multi-directional suture retentionstrength of unreinforced and reinforced tissue grafts before and after21 days incubation in 1×PBS (pH=7.4) at 37° C.;

FIG. 8 is a graph illustrating load-displacement plots of unreinforcedtissue grafts and tissue grafts reinforced using different stitchdesigns and tested using a multi-directional ball burst test;

FIG. 9 is a graph illustrating load displacement plots of unreinforcedtissue grafts and tissue grafts reinforced using different stitchdesigns tested in uniaxial tension with side constraint;

FIG. 10 is a graph illustrating the uniaxial suture retention strengthof unreinforced and reinforced tissue grafts using a peripheral stitchdesign;

FIG. 11 is a graph illustrating the multi-directional suture retentionstrength of unreinforced and reinforced tissue grafts using a peripheralstitch design;

FIG. 12 is a graph illustrating the uniaxial suture retention strengthof unreinforced and reinforced tissue grafts using a rectangularcross-hatch stitch design;

FIG. 13 is a graph illustrating the cyclic elongation during uniaxialfatigue loading of unreinforced and reinforced tissue grafts using arectangular cross-hatch stitch design;

FIG. 14 is a schematic illustration of bone fixation methods forunreinforced and reinforced grafts;

FIG. 15 is a graph illustrating the failure load of unreinforced andreinforced tissue grafts for various bone fixation methods;

FIG. 16 is a graph illustrating the cyclic creep of unreinforced andreinforced tissue grafts for various bone fixation methods;

FIG. 17 is a graph illustrating the uniaxial load-displacement curve ofreinforced tissue grafts for various bone fixation methods;

FIG. 18 is a graph illustrating the uniaxial suture retention strengthof unreinforced tissue grafts and tissue grafts reinforced withresorbable and non-resorbable fibers; and

FIG. 19 is a graph illustrating the uniaxial load-displacement curves oftissue grafts reinforced with resorbable and non-resorbable fibers.

DETAILED DESCRIPTION

The present invention is directed to tissue grafts and, in particular,is directed to a fiber reinforced tissue graft with improved fixationretention properties. The tissue graft can be used to treat a tissuedefect of a subject (e.g., human being), such as a musculoskeletaldefect, or in tendon-to-bone repairs (e.g., rotator cuff injury), orsoft-tissue repairs, such as the repair of lacerated muscles, muscletransfers, or use in tendon reinforcement. The tissue graft may also beused as a bridging material in a subject in the case where the gapbetween a tendon and the associated bone is too large to repairconventionally. The tissue graft can be incorporated between thebone-tendon interface and fixed to the bone and tendon to repair a gapor tear.

The tissue graft in accordance with the present invention includes anextracellular matrix (ECM) patch (or ECM) and a reinforcing means. TheECM can be derived from any mammalian ECM, such as fascia, and inparticular, fascia lata from humans. The ECM can be derived from otherconnective tissue materials, such as dermis as long as the ECM isbiocompatible with the target site or the tissue injury being treated inthe subject or both. The ECM can also be derived, for example, fromother tissues and/or other materials, such as collagen, skin, bone,articular cartilage, meniscus, myocardium, periosteum, artery, vein,stomach, large intestine, small intestine, diaphragm, tendon, ligament,neural tissue, striated muscle, smooth muscle, bladder, ureter,abdominal wall fascia, and combinations thereof.

The ECM used to form the tissue graft may be obtained directly frommammalian tissue (such as an autograft, allograft or xenograft). Thesetissues may be obtained from patients at the time of surgery or acommercial source, such as a tissue bank medical device company. ECMobtained from tissue banks and other commercial sources may be formedusing proprietary processing techniques or modified by additionalprocessing techniques before it is used. In one example, thesetechniques can be used to remove cells and other potentially infectiousagents from the ECM.

The reinforcing means can include any structure or material that isapplied to the ECM, is capable of mitigating tearing of the graft whenthe graft is fixed to tissue being treated, and/or is capable ofincreasing or improving the fixation retention properties of the tissuegraft beyond that which is present in a patch of the ECM alone. Thefixation retention properties can be tailored to increase the graft'sability to remain secured to anatomic structures, such as bone and softtissues, when used to treat a tissue defect. The graft may be secured tothese anatomical structures by, for example, weaving, screws, staples,sutures, pins, rods, other mechanical or chemical fastening means orcombinations thereof. For instance, the graft may be secured to thetreated tissue via different suture configurations, such as, massivecuff, mattress stitching and simple suture and different fixationtechniques, such as, Synthes screw or Biotenodesis screw fixation andsuture anchors with a Krakow stitch.

In one aspect of the invention, the reinforcing means can include athread or strands of fiber(s) that are stitched in a reinforcementpattern in the ECM patch. Fiber stitched in a reinforcement pattern canincrease the fixation properties of the tissue graft, which will resultin a tissue graft having improved mechanical properties for implantationand repair of anatomical defects in a subject. The reinforcement patterncan include any stitch pattern that mitigates tearing and/or improvesthe fixation retention properties of the tissue graft when administeredto a subject being treated. For example, the stitch pattern can includeone or more generally concentric, peripheral or cross-hatched stitchpatterns.

The fiber can enhance the fixation retention of the tissue graft oncestitched into the graft. The fiber can be formed from a biocompatiblematerial that is bioresorbable, biodegradable, or non-resorbable. Theterm bioresorbable is used herein to mean that the material degradesinto components, which may be resorbed by the body and which may befurther biodegradable. Biodegradable materials are capable of beingdegraded by active biological processes, such as enzymatic cleavage.

One example of a biocompatible material that can be used to form thefiber is silk. The silk may include, for example, sericin-free silkfibroin or silk-fibroin modified with a peptide sequence that sequestersgrowth factors in vivo, such as disclosed in U.S. Pat. No. 6,902,932,which is herein incorporated by reference. The fibers can also be formedfrom biodegradable polymers including poly(glycolic acid) (PGA),poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA),poly(ethylene glycol) (PEG), blends thereof, and copolymers thereof. Byway of example, the reinforcing fiber may include a core of PGAsurrounded by a sheath of reinforced PLA fibers. The PGA and PLA may beobtained, for example, from Concordia Fibers in Coventry, R.I. Otherexamples of biocompatible polymers that can be used to form the fiberare resorbable polyesters, such as polyhydroxyalkanoates (PHA), andnon-resorbable fibers, such as polyethylene terephthalate (PET) andultra-high molecular weight polyethylene (UHMWPE). It will beappreciated that the biocompatible fiber can be formed from otherbiocompatible materials, such as other biocompatible materials that aretypically used in forming biocompatible fibers for in vivo medicalapplications.

Regardless of the material used for the fiber of the reinforcing means,the fiber should exhibit a high modulus of elasticity and a failure loadtailored to meet particular design criterion corresponding with in vivostrength requirements of the treated tissue. For example, reinforcedpatches used for the treatment of large and massive rotator cuffs shouldexhibit failure loads of greater than about 250 Newtons (N) at a time ofimplantation, and greater than about 150 N after about one week ofimplantation in vivo. Alternatively, reinforcing patches used for thetreatment of tissues experiencing lower natural loads may be required toexhibit failure loads of about 30 N to about 50 N. It will beunderstood, however, that the fibers, their stitch design (i.e.,reinforcement pattern), or the particular ECM can be tailored to producefailure loads of the fiber-reinforced ECM patch commensurate in scale toany tissue treated within the body.

In an aspect of the invention, the fibers and/or the ECM can bemechanically, chemically or biologically modified to enhance adhesionbetween the fibers and ECM to further secure the fibers to the ECM. Thismodification may occur before or after the fibers are incorporated intothe ECM. This modification may be performed on a portion of orsubstantially all of the stitched fibers or the ECM or both. Duringloading of the tissue graft, the fibers may begin to displace relativeto the ECM and may ultimately completely slip out from the ECM andbecome the primary load bearing components of the reinforced tissueconstruct. It therefore becomes desirable to mitigate or prevent fiberslippage in order to ensure that usage loads are borne by the entiregraft and not just the fibers. Adhesion characteristics of the fiberscan be improved by ablation via ultra-violet (UV) or infrared (IR)light, UV cross-linking or chemical cross-linking, plasma etching, ionetching, coating the fibers with microspheres, application ofbioadhesives or combinations thereof. These treatments can likewise beperformed on the ECM.

In another aspect of the invention, the ECM can be processed to becomedecellularized. Once decellularized, cells can be seeded into thedecellularized ECM that enhance the therapeutic potential of the tissuegraft. For example, the ECM can be seeded with a plurality of progenitorcells that become dispersed in the ECM. Examples of progenitor cells areknown in the art and can include bone marrow-derived progenitor cells,hematopoietic stem cells, endothelial progenitor cells, mesenchymal stemcells, multipotent adult progenitor cells (MAPCs), embryonic stem cells,stromal cells, stem cells, embryonic stem cells, chondrocytes,osteoblasts, and tenocytes. The progenitor cells can be autologous,allogeneic, xenogeneic or a combination thereof. The progenitor cellscan also be genetically modified. Genetically modified cells can includecells that are transfected with an exogenous nucleic acid and thatexpress a polypeptide of interest including, for example, a growthfactor, a transcription factor, a cytokine, and/or a recombinantprotein.

The ECM can additionally or optionally include at least one biologicallyactive molecule dispersed or seeded therein. Any desired biologicallyactive molecule can be selected for impregnating into the ECM. Forexample, the biologically active molecule can include enzymes, hormones,cytokines, colony-stimulating factors, vaccine antigens, antibodies,clotting factors, angiogenesis factors, regulatory proteins,transcription factors, receptors, and structural proteins. Thebiologically active molecule can be chosen based on where themusculoskeletal graft is to be located in the subject or thephysiological requirements of the subject or both. For example, if themusculoskeletal graft is used to repair a tendon, the biologicallyactive molecule which is seeded on or into the ECM can be a growthfactor such as IGF-I, TGF-α, VEGF, bFGF, BMP or combinations thereof.

Optionally, a high-molecular weight (e.g., greater than about 250 kDa)hyaluronic acid (HA) can be incorporated into the tissue graft prior to,during, or after stitching of the fibers into the ECM. When incorporatedinto the tissue graft, HA can potentially inhibit the migration ofinflammatory cells, induce the migration of non-inflammatory cells, andpromote angiogenesis, which would promote integration of the ECM withthe underlying host tissues.

The high-molecular weight HA can be cross-linked within the ECM tomitigate diffusion of the HA from the ECM. Cross-linked,high-molecular-weight HA can be retained in ECM for extended periods invitro. An example of a cross-linked HA material that can be used in thisapplication is prepared by substituting tyramine moieties onto the HAchains and then linking tyramines to form dityramine linkages between HAchains, effectively cross-linking or gelling the HA into the ECM.Examples of dityramine-cross-linked HA composition and chemistry aredisclosed in U.S. Pat. No. 6,982,298 and U.S. Application PublicationsNos. 2004/0147673, 2005/0265959, and 2006/0084759, which are hereinincorporated by reference. The tyramine-substitution rate on the HAmolecules may be about five percent based on available substitutionsites as disclosed in the aforementioned publications.

TS-HA can be impregnated into the ECM, and then immobilized within ECMby cross-linking of the tyramine adducts to form dityramine linkages,thereby producing a cross-linked HA macromolecular network. The TS-HAcan be impregnated into the ECM prior to or after stitching the ECM.

The TS-HA can be used to attach fibronectin functional domains (FNfds)to the ECM in order to further promote healing, cell migration, andanti-inflammatory capabilities. FNfds possess the ability to bindessential growth factors that influence cell recruitment andproliferation (e.g., PDGF-BB and bFGF). The FNfds may, for example,constitute fibronectin peptide “P-12” with a C-terminal tyrosine toallow it to be cross-linked to TS-HA.

One example of a tissue graft in accordance with the present inventionis illustrated in FIG. 1. The tissue graft 10 includes a reinforced ECMpatch or strip that can be used to augment a tendon or muscle repair tobone in, for example, a rotator cuff injury. The tissue graft 10includes an ECM patch 20 and a means 22 for reinforcing the patch.

The patch 20 is illustrated as having a generally rectangular stripshape (e.g., about 5 cm long by about 2 cm wide) although the patch canhave other shapes, such as an elliptical shape, a circular shape, asquare shape, etc. (e.g., FIGS. 2, 3, 4). The patch 20 includes a topsurface 24 and a substantially parallel bottom surface 26 spaced fromthe top surface. A first side 28 and second side 30 connect the topsurface 24 to the bottom surface 26. The first and second sides 28, 30extend generally parallel to one another. The patch 20 further includesa front surface 32 and rear surface 34 which connect the first side 28to the second side 30. The front and rear surfaces 32, 34 extendgenerally parallel to one another.

The reinforcing means 22 can include at least one fiber disposed orprovided within the patch 20 by, for example, conventional stitchingtechniques. By stitching, it is meant that at least one fiber of thereinforcing means 22 is stitched into the patch 20 such that each stitchof the reinforcing means extends between and through both the topsurface 24 and the bottom surface 26 of the patch 20 to securely fastenthe reinforcing means to the patch.

The reinforcing means 22 may exhibit any reinforcement configuration orpattern that increases the fixation properties of the patch 20. One suchconfiguration is illustrated in FIG. 1 in which first and second fibers40, 42 are stitched into the patch 20 in geometrically concentricconfigurations. Additionally or alternatively, the stitch lines of thefibers can be placed further away from the edges of the patch 20 todelay, mitigate, or prevent slipping of the fibers 40, 42 within thepatch 20. Although FIG. 1 illustrates two fibers in a geometricallyconcentric pattern, it will be understood that more or fewer fibers canbe stitched into the patch in a geometrically concentric pattern.Additionally, it will be appreciated that additional fibers can bestitched into the ECM patch 20 in other reinforcement patterns.

As shown in FIG. 1, the first fiber 40 can extend substantially parallelto, and be spaced inwardly from, the periphery of the patch 20. By wayof example, the first fiber 40 can extend substantially parallel to thefirst and second sides 28, 30 and the front and rear surfaces 32, 34 ofthe patch 20 such that the first fiber 40 exhibits a generallyrectangular configuration. The first fiber 40 can comprise a pluralityof interconnected stitches 41. The ends of the fiber 40 may be stitchedtogether (not shown) to form a continuous stitching construction.

The second fiber 42 can extend substantially parallel to the first fiber40 and be disposed radially inward of the first fiber 40 within thepatch 20. In this configuration, the first and second fibers 40, 42 forma generally geometrically concentric construction in a peripheral doublepass orientation. The second fiber 42 can comprise a plurality ofinterconnected stitches 43. The second fiber can be substantiallyuniformly spaced inward from the first fiber 40 by a gap indicated by“s₁”. The gap s₁ may be, for example, on the order of about 1 mm toabout 3 mm (e.g., about 2 mm), although other spacing configurationswill be understood. It will be appreciated that although the gap s₁between the fibers 40 and 42 is substantially uniform, the gap s₁ mayvary depending on reinforcement pattern in which the fibers 40 and 42are stitched. The ends of the second fiber 42, like first fiber 40, maybe stitched together (not shown) to form a continuous stitchingconstruction.

The first fiber 40 and the second fiber 42 can be stitched in the ECM sothat the number of stitches per inch is, for example, about 10 stitchesper inch to about 20 stitches per inch (e.g., about 15 stitches perinch). Generally, the more stitches per inch, the greater the strengthof the reinforcing means 22 and the fixation retention properties of thetissue graft 10. In some examples, however, it may be desirable to useless stitches per inch to avoid excessive needle penetrations in the ECM20, which may potentially weaken the tissue graft 10.

Other examples of concentric reinforcement stitch patterns orconfigurations are illustrated in FIG. 2 and FIGS. 3A-3C. Theconfigurations in FIG. 2 and FIGS. 3A-3C are similar to theconfiguration of FIG. 1, except that in FIG. 2 the patch 20 issubstantially circular and therefore the reinforcing means 22 isprovided in the patch in a generally circular configuration ororientation. FIG. 2 illustrates one example of a patch 20 that includesgenerally concentric reinforcement means 22 in a peripheral double passorientation. The reinforcement means 22 includes a first fiber 40 thatcomprises a plurality of interconnected stitches 41 and a second fiber42 that comprises a plurality of interconnected stitches 43. The firstfiber 40 can extend substantially parallel to, and be spaced inwardlyfrom, a peripheral surface 33 of the patch 20 such that the first fiberhas a generally circular configuration. The second fiber 42 can extendsubstantially parallel to the first fiber 40 and be disposed radiallyinward of the first fiber within the patch 20. In this configuration,the first and second fibers 40, 42 form a generally concentricconstruction.

FIG. 3A illustrates another example of the reinforcing means 22comprising two concentric patterns 43. Each concentric pattern 43includes a first strand 40 that comprises a plurality of interconnectedstitches 41 and a second strand 42 that comprises a plurality ofinterconnected stitches 43. Each first fiber 40 can extend substantiallyparallel to, and be spaced inwardly from, a peripheral surface 33 of thepatch 20 such that each first fiber has a generally circularconfiguration. Each second fiber 42 can extend substantially parallel tothe first fiber 40 and be disposed radially inward of the first fiberwithin the patch 20. In this configuration, each pair of first andsecond fibers 40, 42 form a generally concentric construction. Althoughthe two concentric patterns 43 are illustrated as being substantiallysemi-circular, it will be understood that each concentric pattern mayexhibit alternative constructions such as, for example, rectangular(e.g., in a two rectangle double pass orientation), elliptical,triangular or combinations thereof within the spirit of the presentinvention.

FIG. 3B illustrates another example of the reinforcing means 22comprising three concentric patterns 43. Each concentric pattern 43comprises a first strand 40 comprising a plurality of interconnectedstitches 41 and a second strand 42 comprising a plurality ofinterconnected stitches 43. Each first fiber 40 can extend substantiallyparallel to, and be spaced inwardly from, a peripheral surface 33 of thepatch 20 such that each first fiber has a generally circularconfiguration. Each second fiber 42 can extend substantially parallel tothe first fiber 40 and be disposed radially inward of the first fiberwithin the patch 20. In this configuration, each pair of first andsecond fibers 40, 42 form a generally concentric construction. It willbe understood that each concentric pattern may exhibit any constructionssuch as, for example, rectangular (e.g., in a three rectangle doublepass orientation), elliptical, triangular, semi-circular, circular orcombinations thereof within the spirit of the present invention.

FIG. 3C illustrates yet another example of a reinforcing meansreinforcing means 22 that includes a plurality of first strands 40,which comprise a plurality of interconnected stitches 41 but without orfree of concentric second strands 42. In particular, the first strands40 may comprise four substantially parallel and elliptical discretefirst strands. Although the four first strands 40 are illustrated asbeing substantially elliptical, it will be understood that each firststrands may exhibit alternative constructions such as, for example,rectangular (e.g., in a four rectangle single pass orientation),semi-circular, circular, triangular or combinations thereof within thespirit of the present invention. It will also be understood that one ormore of the first strands could have a geometrically concentric patternwith a second strand within the spirit of the present invention.

FIG. 4A is a schematic illustration of a tissue graft 10 that includesan ECM patch 20 and a reinforcing means 22 in accordance with anotherexample of the invention. The reinforcing means 22 includes a pluralityof first fibers 40 and a plurality of second fibers 42 stitched in across-hatched pattern across the patch 20 and between the first andsecond sides 28, 30 and the front and rear surfaces 32, 34. AlthoughFIG. 4A illustrates six first fibers 40 and eight second fibers 42, itis understood that more or less of each fiber may be utilized inaccordance with the present invention. The first fibers 40 can extend ina first direction, indicated by “d₁”, across the top surface 24 of thepatch 20 from the first side 28 to the second side 30. Each of the firstfibers 40 can extend parallel to one another and be spaced apart by agap indicated by “s₁”. The gap s₁ may be, for example, on the order ofabout 1 mm to about 3 mm, although other spacing configurations will beunderstood. The gap s₁ may be uniform or may vary between first fibers40.

The second fibers 42 can extend in a second direction, indicated by“d₂”, across the top surface 24 of the patch 20 from the front surface32 to the rear surface 34. The directions “d₁” and “d₂” in which thefirst and second fibers 40, 42 extend may be configured such that thefirst fibers and the second fibers are oriented perpendicular to eachother. Each of the second fibers 42 can extend parallel to one anotherand be spaced apart by a gap indicated by “s₂”. The gap s₂ may be, forexample, on the order of about 1 mm to about 3 mm, although the gap canhave other spacing configurations. The gap s₂ may be uniform or may varybetween second fibers 42. The second fibers 42 are disposed in anoverlying fashion relative to the first fibers 40 such that the firstfibers are disposed between the top surface 24 of the patch 20 and thesecond fibers. The second fibers 42, however, could alternatively bedisposed between the top surface 24 of the patch 20 and the first fibers40.

FIG. 4B illustrates that the reinforcing means 22 comprises a pluralityof first fibers 40 and a plurality of second fibers 42 stitched in across-hatched pattern across the patch 20 and between the first andsecond sides 28, 30 and the front and rear surface 32, 34. Although FIG.4B illustrates four first fibers 40 and four second fibers 42, it isunderstood that more or less of each fiber may be utilized in accordancewith the present invention. Each of the first fibers 40 extends from thefirst side 28 to the second side 30 of the patch 20. Each of the secondfibers 42 extends from the front surface 32 to the rear surface 34 ofthe patch 20. The ends of the first fibers 40 and the ends of the secondfibers 42, respectively, may be stitched together (not shown) to form acontinuous stitching construction.

The second fibers 42 are disposed in an overlying fashion relative tothe first fibers 40 such that the first fibers are disposed between thetop surface 24 of the patch 20 and the second fibers. The second fibers42, however, could alternatively be disposed between the top surface 24of the patch 20 and the first fibers 40. Although the first and secondfibers 40, 42 are illustrated as having a substantially rectangularshape (e.g., a rectangular cross-hatch orientation), it will beunderstood that the first fiber 40 and/or the second fiber 42 mayexhibit alternative constructions such as elliptical, semi-circular,circular, triangular or combinations thereof within the spirit of thepresent invention.

The tissue graft of the present invention can be used in tissueengineering and musculoskeletal repair, such as rotator cuff repair, butis not restricted to musculoskeletal applications. The graft may beadministered to a subject to mechanically and biologically augment therepair by placing it over a tendon-bone repair or interpositionallygrafting a rotator cuff tendon defect. It will be appreciated thatsimilar methods and materials as described herein could also be adaptedto other tendon-to-bone repairs, soft-tissue repairs, such as the repairof lacerated muscles, muscle transfers, spanning a large muscle defect,or use in tendon reinforcement. These applications require secureconnections between the graft 10 and the anatomical site. Fixationtechniques to soft tissue using conventional or novel suture methods, orthe Pulvertaft weave technique (M. Post, J Shoulder Elbow Surg 1995;4:1-9) may be utilized in accordance with the present invention.Fixation techniques to bone using conventional or novel suture methods,anchors, screws, or plates may be utilized in accordance with thepresent invention.

The graft 10 may also serve as a delivery platform for the futureinvestigation of any number of biologic strategies aimed to enhancemuscular skeletal repair, e.g., rotator cuff healing. Furthermore, thegraft 10 could be effective for other needs in the field of surgicalreconstruction, including ligament reconstruction, bowel and bladderreconstruction, abdominal wall repair, and tendon reconstruction in thesetting of post-surgical repair failure, trauma and segmental defects.The following examples are illustrative of the principles and practiceof this invention. Numerous additional embodiments within the scope andspirit of the invention will become apparent to those skilled in theart.

Example 1

In this example, two groups were investigated: Group I-Control—Native(unreinforced) fascia and Group II-Experimental—reinforced fascia. Thefascia was reinforced using a biodegradable polymer braids as thereinforcing material. Polymer braids used were made of poly lactic acid(PLA) and poly glycolic acid (PGA). PLA and PGA are the most widelyresearched polymers in the field of tissue engineering. Since PGAdegrades more rapidly than PLA, the present example uses polymer braidshaving PGA as a core and a combination of PLA/PGA as a sheath (FIG. 5A).Two tests were used to verify the efficacy of stitching as a method ofreinforcement with polymer braids, namely, uniaxial tension test andmulti directional loading using a modified Ball burst test.

All allograft human fascia lata were obtained from the MusculoskeletalTransplant Foundation in Edison, N.J. (donor age 18-55 years). All PGAand PLA braids used for reinforcing the fascia were obtained fromConcordia Fibers, Coventry, R.I.

Uniaxial Suture Retention Test

A sample size of (n=10) unreinforced patches were used as the control.Each consisted of 2 cm wide×5 cm long strips of ECM hydrated for 20minutes in saline solution and maintained at room temperature. Theunreinforced patches were tested with either one mattress (n=5) or oneMason Allen suture (n=5) placed 5 mm away from the 2 cm wide edge. Atemplate was created to assure uniformity in the placement of thesutures. The two sutures were tied over a tubular rod mounted on thecross head of a MTS 5543 table top system.

A sample size of (n=7) reinforced fascia were used to test the abilityof stitching with two types of PLA/PGA braid to improve the sutureretention strength over unreinforced fascia. This sample size wasselected due to the preliminary nature of the study. Each reinforcedpatch consisted of fascia 2 cm wide×5 cm long reinforced with a polymerbraid having a sheath of 4PLA and 4PGA with (n=2) and without (n=5) a2PGA core. The reinforced patches were tested with two simple suturesplaced 5 mm away from the 2 cm wide edge. The suture retention load wasdefined as the maximum load attained by the specimen.

The results of this test are illustrated in FIG. 5 b. FIG. 5 b showsthat stitching as a reinforcement method has the ability to increase thesuture retention strength of fascia. Additionally, the presence of a PGAcore in the polymer braid positively impacts the reinforcement byincreasing the suture retention strength of the tissue. The sutureretention loads obtained with the polymer braids having no PGA core (84N) were about half the suture retention loads attained with polymerbraids having a PGA core (146 N).

Ball Burst Test

Depending on the size, location, and chronicity of the tear in vivo, thegraft may be subjected to biaxial tensile forces. Therefore, experimentsusing a modified Ball Burst test, inspired from the ASTM D3787 BallBurst test standard used to determine the bursting resistance of knittedfabrics and goods under multi-axial forces, were used to quantify thesuture retention strength. In particular, 4 cm diameter discs of theunreinforced and reinforced fascia were hydrated for 20 minutes insaline at room temperature. The hydrated specimens were then sutured toa stainless steel ring (5 cm outer diameter) in a simple sutureconfiguration at 1 cm increments using No. 2 Fiberwire (ArthrexCorporation, Naples, Fla.). The fascia-steel construct was then mountedon a specially designed fixture, which was then mounted on the base ofthe MTS1321 system. A polished stainless steel ball having a 1″ diameterwas attached to the cross head of the MTS system and pushed through thespecimen at a constant distraction rate of 6 mm/min. The sutureretention load was noted as the maximum load attained by the specimenprior to a 10% or more drop in the peak load.

The results of the Ball Burst test are illustrated in FIG. 6. FIG. 6shows that even with the Ball Burst test, which is more rigorous thanthe uniaxial test in that it subjects the specimen to multi-directionalloading, the reinforced fascia construct has suture retention strengthabout 3 to about 4 times greater than unreinforced fascia. FIG. 6 alsoillustrates that the amount of PGA in the polymer braid directly affectsthe suture retention strength. This result was expected, as PGA hashigher tensile strength compared to PLA.

Example 2 In Vitro Degradation Study

Fascia discs having a diameter of 4 cm were stitched along the peripheryusing PLA, PGA, and PE polymer braids (n=6 per group). Three specimensper group were allocated to time zero testing and three were subjectedto in vitro degradation. For in vitro degradation, the specimens wereput in individual beakers containing 100 mL of 1×PBS (pH=7.4) andimmersed in a water bath maintained at 37° C.

The 1×PBS solution was checked every day for any signs of contaminationand the solution was changed every other day so as to maintain aconstant pH of 7.4 throughout the study. At the end of the 21 days thespecimens were removed and sutured to a stainless steel ring in simplesuture configuration at 1 cm intervals. The suture retention loads ofthe two groups, time zero and 21 days, were quantified using themodified ball burst test. Failure testing included 10 cycles ofpreconditioning form 5-15 N at 0.25 Hz followed by load to failure at 30mm/min.

Suture retention load of fascia stitched with the three polymer braidsat the two time points, time zero and 21 days, is shown in FIG. 7. FIG.7 shows that the suture retention loads for fascia stitched with thethree polymer braids is significantly higher than native fascia at bothtime zero and 21 days. The suture retention load of stitched fascia wasnot significantly different within a group as a function of time, orbetween groups at either time point. The data show that stitched fasciasignificantly increases the suture retention load of fascia and theincrease is maintained for at least 21 days in simulated in vivoconditions.

Example 3 Design Configurations

4×4 cm pieces of fascia were stitched using 2-0 commercial silk suture(Harvard Apparatus, Holliston, Mass.) using five stitchconfigurations: 1) peripheral double pass; 2) 2 rectangle double pass;3) 3 rectangle double pass; 4) 4 rectangle double pass; and 5)rectangular cross-hatch. The samples were tested using the previouslydescribed modified ball burst test and a pseudo side constraint test.For the pseudo side constraint test, the sample was constrained to astainless steel ring using simple suture configurations and wasdistracted in uniaxial tension to failure at 30 mm/min.

The results are illustrates in FIGS. 8-9. Using both test methods, therectangular cross-hatch stitch pattern had the highest suture retentionloads compared to other stitch configurations investigated. These datashow that the rectangular cross-hatch stitch pattern will make themechanical performance of fascia suitable for large to massive rotatorcuff tendon repairs.

Example 4

A polymer braid having a 6PLA sheath with 2PGA core (Concordia Medical,Coventry, R.I.) was stitched into strips and patches of human fascialata ECM. To model in vivo physiologic loading, the suture retentionloads were quantified using three different tests: unidirectional pull(FIG. 10), modified ball burst (FIG. 11), and tension with sideconstraint (FIGS. 12-13). For all tests, load was applied to the samplesusing #2 Fiberwire simple sutures (Arthrex Corporation, Naples, Fla.).

Specimens were subjected to failure testing using all three types oftests. Failure testing included 10 cycles of pre-conditioning from 5-15Nat 0.25 Hz followed by constant rate distraction to failure at 30mm/min. As well, samples were subjected to cyclic fatigue testing (5-150N, 5000 cycles at 0.25 Hz in a saline bath) using the tension with sideconstraint test.

The results indicate that reinforcing fascia lata ECM with abiodegradable polymer significantly increases its suture retentionstrength to physiologically relevant loads (>100 N) (FIGS. 10-12).Further, the reinforced patch can resist cyclic fatigue loading atphysiologically relevant loads (5-150 N) for up to 5000 cycles (FIG.13). Hence, reinforced fascia may provide a natural, strong, andmechanically robust scaffold for bridging tendon or muscle defects.

Example 5 Bone Fixation Method

16×1.5 cm pieces of fascia were stitched using a peripheral double passconfiguration using 6PGA and 6PLA polymer braids. Reinforced fascia wasrepaired to Sawbones using the following fixation techniques: 1) Krakowstitch with post (models suture anchor) (FIG. 14A, n=2); 2) Screw andwasher fixation (FIG. 14B, n=2); and Biotenodesis interference screw(FIG. 14C, n=2). The samples were tested 100 cycles, 5-50 N at 0.25 Hzand then loaded to failure at 30 mm/min.

The results are summarized in FIGS. 15-17. Failure load is higher inreinforced fascia than native fascia with all bone fixation methods(FIG. 15), and failure load is not different between fixation methods orfiber types tested (FIG. 15). Krakow stitch fixation allows more cycliccreep than the other methods (FIG. 16). Representative load-displacementcurves for the failure portion of the test for samples reinforced withPGA fiber are shown in FIG. 17. Biotenodesis screw fixation proved to bethe stiffest of the three methods during the failure portion of the test(FIG. 17).

Example 6 Suture Retention Test with Non Resorbable Fibers

The studies were conducted to compare the suture retention load offascia reinforced with non-resorbable polyesters, sizes 2-0 and 3-0polyethylene terephthalate (PET) braided suture (Ashaway Twin Mfg. Co.,RI) and ForceFiber™ UHMWPE braided suture (Teleflex Medical, Mass.).Suture retention loads were quantified using the uniaxial sutureretention test and compared to fascia reinforced with size 2-0 custombraided PLA suture (Concordia Fibers, R.I.).

Each 2×5 cm strip was provided with an inner stitch line placed 5 mmaway from the edge of the tissue and an outer stitch line placed 3 mmfrom the edge of the tissue. A template was created to assure uniformityin the placement of the sutures.

The suture retention load, defined as the maximum load attained by thespecimen was quantified using a standard pull to failure test with onesimple suture. The specimen was preloaded to 2 N and then distracted tofailure at a rate of 30 mm/min.

The results of this test are illustrated in FIG. 18. FIG. 18 shows thatstitching as a reinforcement method has the ability to increase thesuture retention strength of fascia (compare to unreinforced fasciashown in FIG. 10). Even though different polymer braids have been used,the table clearly indicates an increase in suture retention properties,irrespective of the polymer braid used as the reinforcing material.Additionally, suture retention strength of the fascia reinforced withFORCE FIBER in both sizes was significantly higher compared to fasciareinforced with the PET braided and PLA braided suture materials. Nosignificant difference, however, in suture retention strength was foundbetween 2-0 (174±39 N) and 3-0 (173±20 N) FORCE FIBER reinforced fascia.Further, the 2-0 PET reinforced fascia (129±8 N) had a significantlyhigher suture retention load compared to the 3-0 PET reinforced fascia(87±5 N) and the PLA reinforced fascia (106±9 N).

Suture Displacement Test

FIG. 19 gives the average load displacement plots (LD) of 2×5 cm stripsof fascia reinforced with 2-0 and 3-0 sized PET braided suture (AshawayTwin Mfg. Co., RI) and FORCE FIBER UHMWPE braided suture (TeleflexMedical, Mass.). The LD plot was also generated for fascia reinforcedwith 6PLA.

The LD plots for fascia reinforced with different suture materials isessentially the same until 5 mm of displacement. Visual inspectionsuggests that initially both the fibers and fascia matrix are loaded asthe fibers slip through the fascia matrix. After about 5 mm ofdisplacement, however, the fibers completely slip out from the fasciamatrix and become the primary load bearing components of the reinforcedfascia construct.

The complete slippage of fibers from the fascia matrix corresponds tothe initial placement of the stitch lines at 5-10 mm from the edge ofthe fascia patch. After the fiber has completely slipped from thefascia, the maximum load attained by the reinforced fascia constructdepends on the ultimate tensile strength and knot breaking strength ofthe respective fibers used to reinforce the fascia.

For the PET samples, slipping of the fiber at the fiber-fascia interfacewas followed by breaking of the stitched fiber loop at displacementsgreater than 5 mm. The FORCE FIBER samples failed when the inner stitchline unraveled in the direction of loading together with pulling alongthe stitching lines and breaking of the stitched loops.

It may be concluded from the LD plots that 2-0 FORCE FIBER reinforcedfascia is stiffer than fascia reinforced with other suture materials.The large error bars seen in FIG. 19 for the 2-0 FORCE FIBER reinforcedfascia are due to the divergent behavior of one of the four testedspecimens.

1. A biocompatible tissue graft comprising: an extracellular matrixpatch; and a means for reinforcing the patch to mitigate tearing and/orimprove fixation retention of the patch, wherein the reinforcing meanscomprises at least one fiber stitched into the patch in a reinforcementpattern.
 2. (canceled)
 3. The tissue graft of claim 1, wherein thereinforcing means comprises fibers stitched into the patch in aconcentric pattern.
 4. The tissue graft of claim 3, wherein thereinforcing means comprises multiple concentric patterns of fibersstitched into the patch.
 5. The tissue graft of claim 1, wherein thereinforcing means comprises fibers stitched into the patch in across-hatched configuration.
 6. The tissue graft of claim 1, wherein thefiber is selected from the group consisting of silk, sericin free silk,modified silk fibroins, polyesters like polyglycolic acid (PGA),polylactic acid (PLA), polylactic-co-glycolic acid (PLGA),polyethyleneglycol (PEG), polyhydroxyalkanoates (PHA), polyethyleneterephthalate (PET), polyethylene (PE), ultra-high molecular weightpolyethylene (UHMWPE), blends thereof, and copolymers thereof.
 7. Thetissue graft of claim 1, wherein at least one of the patch and the fiberis modified to improve adhesion between the patch and the fiber.
 8. Thetissue graft of claim 1, wherein the patch is decellularized.
 9. Thetissue graft of claim 1, wherein the patch further comprises at leastone progenitor cell.
 10. The tissue graft of claim 1, wherein the patchfurther comprises at least one biologically active molecule selectedfrom the group consisting of enzymes, hormones, cytokines,colony-stimulating factors, vaccine antigens, antibodies, clottingfactors, angiogenesis factors, regulatory proteins, transcriptionfactors, receptors, and structural proteins and combinations thereof.11. A biocompatible tissue graft comprising: a fascia patch; and atleast one fiber stitched into the patch in a reinforcement pattern tomitigate tearing and/or improve fixation retention of the patch.
 12. Thetissue graft of claim 11, wherein the reinforcing means comprises fibersstitched into the patch in a concentric pattern.
 13. The tissue graft ofclaim 12, wherein the reinforcing means comprises multiple concentricpatterns of fibers stitched into the patch.
 14. The tissue graft ofclaim 11, wherein the reinforcing means comprises fibers stitched intothe patch in a cross-hatched configuration.
 15. The tissue graft ofclaim 11, wherein the fiber is selected from the group consisting ofsilk, sericin free silk, modified silk fibroins, polyesters likepolyglycolic acid (PGA), polylactic acid (PLA), polylactic-co-glycolicacid (PLGA), polyethyleneglycol (PEG), polyhydroxyalkanoates (PHA),polyethylene terephthalate (PET), polyethylene (PE), ultra-highmolecular weight polyethylene (UHMWPE), blends thereof, and copolymersthereof.
 16. The tissue graft of claim 11, wherein at least one of thepatch and the fiber is modified to improve adhesion between the patchand the fiber.
 17. The tissue graft of claim 11, wherein the patch isdecellularized.
 18. The tissue graft of claim 11, wherein the patchfurther comprises at least one progenitor cell.
 19. The tissue graft ofclaim 11, wherein the patch further comprises at least one biologicallyactive molecule selected from the group consisting of enzymes, hormones,cytokines, colony-stimulating factors, vaccine antigens, antibodies,clotting factors, angiogenesis factors, regulatory proteins,transcription factors, receptors, and structural proteins. 20-29.(canceled)
 30. A method for repairing tissue in a subject comprising:administering to the tissue a tissue graft comprising an extracellularmatrix patch and at least one fiber stitched into the patch to mitigatetearing and/or improve fixation retention of the patch.
 31. The methodof claim 30, wherein the step of administering the tissue graftcomprises securing the tissue graft in or on recipient tissue.
 32. Themethod of claim 31, wherein the step of securing the tissue graft in oron recipient tissue comprises suturing, stitching, or screwing.